| 名稱 | GSK-1070916 |
| 描述 | GSK-1070916 (GSK-1070916A) is a reversible and ATP-competitive inhibitor of Aurora B/C with IC50 of 3.5 nM/6.5 nM. It displays >100-fold selectivity against the closely related Aurora A-TPX2 complex. Phase 1. |
| 細(xì)胞實(shí)驗(yàn) | Cells are plated in 96-well plates in the recommended growth media and incubated at 37 °C in 5% CO2 overnight. The following day, the cells are treated with serial dilutions of GSK1070916. At this time, one set of cells is treated with CellTiter-Glo for a time equal to 0 (T = 0) measurement. Following a 6- to 7-d incubation with compound, cell proliferation is measured using the CellTiter-Glo reagent according to the manufacture's recommended protocol. As inhibition of Aurora B induces endomitosis, the degree of which differs depending on the cell type, an extended compound treatment time is required to accurately reflect the effects on cell viability across a large panel of cell lines. For analysis of cell viability, values from wells with no cells are subtracted for background correction and the data plotted as a percent of the DMSO-treated control samples using Microsoft Excel XLfit4 software. The EC50 values represent the concentration of GSK1070916 where 50% maximal effect is observed(Only for Reference) |
| 激酶實(shí)驗(yàn) | Kinase Assay: The ability of GSK1070916 to inhibit the Aurora enzymes is measured using in vivo kinase assays. The assays measure the ability of Aurora A, Aurora B and Aurora C to phosphorylate a synthetic peptide substrate. Biotin-Ahx-RARRRLSFFFFAKKK-NH2 is used for the Aurora A–TPX2 LEADseekerTM assay and 5FAM-PKAtide is used for the IMAPTM assay for all three Aurora kinases. To take into account time-dependent inhibition of Aurora enzymes, Aurora A–TPX2, Aurora B–INCENP and Aurora C–INCENP are incubated with GSK1070916 at various concentrations for 30 min before the reactions are initiated with the addition of substrates. For the Aurora A LEADseekerTM assay, final assay conditions are 0.5 nM Aurora A–TPX2, 1 μM peptide substrate, 6 mM MgCl2, 1.5 μM ATP, 0.003 μCi/μL [γ-33P] ATP in 50 mM Hepes, pH 7.2, 0.15 mg/mL BSA, 0.01% Tween-20, 5 mM DTT and 25 mM KCl. The reactions are incubated at room temperature (25 °C) for 120 min and terminated by the addition of LEADseekerTM beads in PBS containing EDTA (final concentration 2 mg/mL beads and 25 mM EDTA). The plates are then sealed, and the beads are allowed to settle overnight. Product formation is quantified using a Viewlux Imager. For the IMAPTM assays, Aurora A–TPX2 (final concentration 1 nM), Aurora B–INCENP (final concentration 2 nM) or Aurora C–INCENP (final concentration 2.5 nM) is added to the compound-containing plates in 5 μL of buffer (25 mM Hepes, pH 7.2, for Aurora A, 25 mM Hepes, pH 7.5, for Aurora B and 20 mM Hepes, pH 7.2, for Aurora C) containing 0.15 mg/mL BSA, 0.01% Tween 20 and 25 mM NaCl. This mixture is incubated at room temperature for 30 min. To start the reaction, 5 μL of a substrate solution is added containing the same Hepes buffer as used for the pre-incubation, 25 mM NaCl, MgCl2 (2, 4 and 4 mM for Aurora A, B and C respectively), DTT (4, 4 and 2 mM for Aurora A, B and C respectively), ATP (4, 4 and 10 μM for Aurora A, B and C respectively), 200 nM 5FAM-PKAtide, 0.01% Tween 20 and 0.15 mg/mL BSA. The reactions are incubated at room temperature for 120 min for Aurora A and B and 60 min for Aurora C. These reactions are then terminated by the addition of 10 μL of 1:500 (1:600 for Aurora C) Progressive Binding Reagent in 95% Progressive Binding Buffer A and 5% Progressive Binding Buffer B. Plates are incubated at room temperature for approx. 90–120 min (time allowed for equilibrium to be reached). Plates are read in a Molecular Devices Analyst plate reader in fluorescence polarization mode. |
| 體外活性 | GSK1070916 對 Aurora B和Aurora C的 Ki值為 0.38 nM和1.5 nM。針對Aurora B和C的抑制作用是時(shí)間依賴的，其酶-抑制劑解離半壽命分別>480分鐘和270分鐘。此外，GSK1070916還是一種與ATP競爭的抑制劑。[1]用GSK1070916處理的人類腫瘤細(xì)胞表現(xiàn)出劑量依賴性地抑制Histone H3第10絲氨酸的磷酸化，這是Aurora B的特異性底物。GSK1070916還能夠抑制超過100種跨越廣泛腫瘤類型細(xì)胞系的腫瘤細(xì)胞增殖，EC50值均<10 nM，中值EC50為8 nM。盡管GSK1070916對增殖細(xì)胞具有強(qiáng)大活性，但在原代、非分裂的正常人靜脈內(nèi)皮細(xì)胞中觀察到其活性有顯著變化。此外，GSK1070916處理的細(xì)胞并不在有絲分裂中停滯，而是失敗分裂成為多倍體，最終導(dǎo)致凋亡。[2]在另一項(xiàng)研究中，還報(bào)告了高染色體數(shù)與對Aurora B和C抑制的抵抗性相關(guān)，這表明具有繞過高倍體檢查點(diǎn)機(jī)制的細(xì)胞對GSK1070916具有抵抗性。[3] |
| 體內(nèi)活性 | GSK1070916（25、50或100 mg/kg）在小鼠中顯示出對Aurora B特異性底物磷酸化的劑量依賴性抑制作用。該化合物在包括乳腺癌、結(jié)腸癌、肺癌和兩種白血病模型在內(nèi)的10種人類腫瘤異種移植模型中展示了抗腫瘤效果。[2] |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 102 mg/mL (200.93 mM), Sonication is recommended.
Ethanol : 8 mg/mL (15.76 mM), Sonication is recommended.
H2O : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | Tie-2 | Tie2 | SIK | Inhibitor | inhibit | GSK-1070916 | GSK 1070916 | FLT1 | AuroraKinase | Aurora Kinase | Aurora C-INCENP | Aurora B-INCENP | Apoptosis |
| 相關(guān)產(chǎn)品 | Chitosan oligosaccharide | L-Glutamic acid | Cysteamine hydrochloride | Alginic acid | Flubendazole | 5-Fluorouracil | Stavudine | Dextran sulfate sodium salt (MW 4500-5500) | Tributyrin | L-Ascorbic acid sodium salt | L-Ascorbic acid | Sodium 4-phenylbutyrate |
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